The field of the present invention broadly relates to retaining nucleic acid integrity in order that such nucleic acid is amenable to certain diagnostic processes, such as hybridization and amplification. More specifically, the present invention relates to the use of compositions that cause an inhibitory affect on proteolytic agents and/or nucleic acid degradative agents.
Diagnostic processes that utilize nucleic acid molecules include nucleic acid probe hybridization to determine the presence and/or amount of a target nucleic acid, nucleic acid primer hybridization for nucleic acid amplification processes and enzymatic activity including nucleic acid extension, nicking and/or cleavage. Nucleic acid amplification processes such as strand displacement amplification (SDA), polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA), transcription mediated amplification (TMA) and others are used to create multiple copies of a particular nucleic acid sequence(s) of interest (target sequence) which is present in lesser copy number in a sample.
A known method for inactivating DNases and proteolytic enzymes is to heat a sample at 100° C. for fifteen minutes (see, for example, Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Other methods for inactivating proteolytic enzymes use chaotropic agents, such as guanidinium thiocyanate or sodium thiocyanate, to denature these enzymatic proteins (see, for example, U.S. Pat. No. 4,843,155, incorporated herein by reference). In addition to inactivating nucleases and proteolytic enzymes, chaotropic agents are also known to promote lysis of cell walls in a wide range of biological materials (U.S. Pat. No. 5,234,809, incorporated herein by reference). The use of chaotropic agents to promote inactivation of proteases or nucleases should thus be avoided when preservation of cell walls is also desired.
A more general description of a method for preserving nucleic acids is provided by PCT Publication WO 00/50640 to Exact Laboratories wherein the use of EDTA and EGTA as nuclease inhibitors in methods for extracting DNA from exfoliated human epithelial cells in stool samples is disclosed. Also, Kyoto Ikagaku Kenkyusho: KK own two Japanese patent disclosures relating to methods for preserving cells in urine. In JP 04118557 A2, the use of citric acid as a buffering agent in addition to EDTA as an antibacterial drug is disclosed, and JP 05249104 A2 discloses the use of sodium fluoride as a fluorine compound in addition to citric acid and EDTA. Sierra, Diagnostics in PCT Publication WO 99/29904 disclose the use of ethylenediaminetetraacetic acid (EDTA), ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), or salts thereof in conjunction with at least one of the “chelator enhancing components” lithium chloride, guanidine, sodium salicylate, sodium perchlorate or sodium thiocyanate. Lithium chloride, guanidine, and sodium thiocyanate are known chaotropes to those skilled in the art. Effective concentrations of chelating enhancing components range from about 0.1 M to about 2.0 M.
Many of these methods are time consuming, labor intensive and/or have cumbersome safety requirements associated therewith. Another problem with methods that utilize relatively severe processing steps or conditions is the loss of some target nucleic acid sequence. Despite the ability of nucleic acid amplification processes to make multiple copies of target sequence (amplicons) from very few original targets, amplification efficiency and detection ability are improved if there are greater numbers of original targets in the sample. The greater detection ability can be very important when processing particularly difficult to detect samples such as acid fast Bacillus (AFB) smear negative Mycobacterium tuberculosis samples.
Another common problem with samples to be subjected to a molecular diagnostic process is the stability of the sample over time. Stability of the sample becomes more important when samples are taken at one location, but are then transported to another location such as a centralized laboratory for molecular diagnostic processing.
For example, many clinically relevant organisms do not maintain their integrity in urine samples and vaginal and cervical swabs for more than about twenty-four (24) hours at room temperature. Thus, refrigeration of such samples during transport to centralized laboratories and/or during storage has become a necessity. One analyte that is commonly tested from urine samples and swabs and is notoriously unstable in samples stored at room temperature is Neisseria gonorrhoeae. 